- Volume 1, Issue 4, 2015

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.

Large-scale recombination events have led to the emergence of epidemic clones of several major bacterial pathogens. However, the functional impact of the recombination on clonal success is not understood. Here, we identified a novel widespread hybrid clone (ST71) of livestock-associated Staphylococcus aureus that evolved from an ancestor belonging to the major bovine lineage CC97, through multiple large-scale recombination events with other S. aureus lineages occupying the same ruminant niche. The recombination events, affecting a 329 kb region of the chromosome spanning the origin of replication, resulted in allele replacement and loss or gain of an array of genes influencing host–pathogen interactions. Of note, molecular functional analyses revealed that the ST71 hybrid clone has acquired multiple novel pathogenic traits associated with acquired and innate immune evasion and bovine extracellular matrix adherence. These findings provide a paradigm for the impact of large-scale recombination events on the rapid evolution of bacterial pathogens within defined ecological niches.

Uropathogenic Escherchia coli (UPEC) is the causative agent of urinary tract infections. Nitric oxide (NO) is a toxic water-soluble gas that is encountered by UPEC in the urinary tract. Therefore, UPEC probably requires mechanisms to detoxify NO in the host environment. Thus far, flavohaemoglobin (Hmp), an NO denitrosylase, is the only demonstrated NO detoxification system in UPEC. Here we show that, in E. coli strain CFT073, the NADH-dependent NO reductase flavorubredoxin (FlRd) also plays a major role in NO scavenging. We generated a mutant that lacks all known and candidate NO detoxification pathways (Hmp, FlRd and the respiratory nitrite reductase, NrfA). When grown and assayed anaerobically, this mutant expresses an NO-inducible NO scavenging activity, pointing to the existence of a novel detoxification mechanism. Expression of this activity is inducible by both NO and nitrate, and the enzyme is membrane-associated. Genome-wide transcriptional profiling of UPEC grown under anaerobic conditions in the presence of nitrate (as a source of NO) highlighted various aspects of the response of the pathogen to nitrate and NO. Several virulence-associated genes are upregulated, suggesting that host-derived NO is a potential regulator of UPEC virulence. Chromatin immunoprecipitation and sequencing was used to evaluate the NsrR regulon in CFT073. We identified 49 NsrR binding sites in promoter regions in the CFT073 genome, 29 of which were not previously identified in E. coli K-12. NsrR may regulate some CFT073 genes that do not have homologues in E. coli K-12.